Construction Of Temperature-Responsive Hydrogel Adjuvant Based On Sodium Alginate And Evaluation Of Its Immune Effect

Adjuvants are an important part of the vaccine to promote the immune effect,which can enhance the strength and duration of the immune response,and even affect the type of immune response.At present,all kinds of vaccines still need excellent adjuvants,and exploring the application potential of new adjuvants is still an important research topic for disease prevention and control.In recent years,due to the good water solubility and safety of natural polysaccharides,it has become a research trend of new adjuvants.However,polysaccharide adjuvants have a short time in the body and are easy to degrade,which makes it difficult to continuously produce efficient immune stimulation.Graphene oxide(GO)has excellent physicochemical properties and immunostimulatory effects,but it has poor biocompatibility and is easy to coagulate in salt solution and serum.In this study,the extracellular polysaccharide(EPS)of lactic acid bacteria WXD233 was non-covalently conjugated to modified graphene oxide(GO)to prepare a new water-soluble nano-adjuvant particle GO-EPS.Compared with GO or EPS alone,GO-EPS has better solubility and antigen sustained release effect,and can assist antigen to produce a sustained and efficient immune response.It has a good adjuvant effect.The specific research content is as follows:The first part is extraction and analysis of extracellular polysaccharide from lactic acid bacteria.The molecular weight of Lactobacillus exopolysaccharide WXD233 ranged from 7.76×10~4 Da to 8.43×10~4Da,and it was mainly composed of galactosyl hydrochloride,glucosamine hydrochloride,galactose,glucose,mannose and glucuronic acid.Congo red staining and XRD spectra showed that the polysaccharide did not contain crystal structure and triple helix structure.In the freeze-dried powder state,EPS is a fine long tubular structure,dissolved in water into a regular sphere with a diameter of about 20-30 nm.Mainly by the→3)-β-D-Glcp NAc-(1→、→4)-β-D-Glcp A-(1→、→6-Man-1→、Man-1→、→2,6-Man-1→、→3-Man-1→、→2-Man-1→this several glycosidic bond.Part two:Preparation and characterization of Lactobacillus exopolysaccharide-GO nanoparticles.The single factor experiment determined that GO:EPS=1:2.5,magnetic stirring for 8h,the system temperature 50℃,GO and EPS binding rate reached the highest,76.8%.Under this condition,the load rate of GO-EPS to OVA reached 110%.Through sample characterization,the graphene lamellae structure was observed to be opened and polysaccharide molecules were distributed on the GO lamellae under transmission electron microscopy.The displacement of characteristic functional groups appeared in FT-IR spectrum,and the Zeta potential and particle size in aqueous solution increased.Atomic force microscopy results showed that the lamellar height of the sample increased with the load of EPS and antigen,which all proved that GO was successfully combined with EPS.Thermogravimetric analysis shows that the nanoparticles have good thermal stability and GO:EPS=1:2.5,which is consistent with the sample preparation ratio.The stability of GO-EPS in vitro was good,and the particle size did not change significantly within 12h in PBS with p H=7.2 and p H=6.0.The third part,GO-EPS immune adjuvant efficacy study.CCK-8 experiment showed that the nano-adjuvant had no significant effect on cell survival rate,and tissue sections were stained with H&E to verify that GO-EPS had no potential toxicity to major organs in mice.Laser confocal microscopy showed that GO-EPS could effectively induce macrophages to phagocytosis antigen and improve the efficiency of antigen presentation.In vivo imaging experiments showed that GO-EPS mode antigen OVA had good slow release effect,and the OVA remained in mice for more than 8 days.Mice were immunized by subcutaneous injection to verify the effect of the adjuvant on the immune response,and OVA specific antibody titers in mice were detected by ELISA.The results showed that,compared with GO,EPS and Alum groups,Go-EPS loaded antigen could significantly increase the antibody titer level in mice,and had a good adjuvant effect.In this study,a new water-soluble nano-adjuvant was constructed by non-covally combining Lactobacillus WXD233 extracellular polysaccharide with GO.It has good biocompatibility,slow release effect and immune stimulation effect,which can effectively induce phagocytosis of macrophages and enhance the level of immune response in vivo.The experimental data and theoretical guidance are provided for exploring and designing new safe and effective polysaccharide GO compliant adjuvants.